The present invention relates to the prevention of Lyme disease in mammals. More specifically, the invention relates to immunogenic formulations and to methods for using them to retard or prevent the development of Lyme disease.
The phrases "Lyme disease" and "Lyme borreliosis" generically denote tick-borne infections caused by the spirochaete Borrelia burgdorferi, representing the most common tick-transmitted disease in both the United States and Europe. Lyme disease is similar to syphilis because it affects many organs, most commonly the skin, nervous system, heart and joints, and because it develops in stages and may become chronic.
Since Lyme disease may mimic other diseases, a need exists for -accurate diagnostic tools, especially in difficult cases where the clinical picture is inconclusive. There is also a need for methods to treat or prevent the disease. Antibiotic therapy may be effective if initiated soon after infection, but prolonged, high dosage treatment is necessary once the disease has progressed. Moreover, antibiotic therapy is not always successful. See Preac Mursic et al., Infection 18: 332-341 (1990). Accordingly, a vaccine to prevent Lyme disease is desirable.
Several antigens of B. burgdorferi are known. Two major outer-surface proteins of B. burgdorferi, ospA (31 kd) and ospB (34 kd), are discussed by Barbour, Clin. Microbiol. Revs. 1:399-414 (1988). OspA is present in most strains but is heterogenous; that is, ospA proteins from different strains may differ in molecular weight and in serological reactivity.
OspB is less widely distributed among strains than ospA but, like ospA, exists in different serological and molecular weight forms. The genes for ospA and ospB, which are plasmid-encoded, have been cloned, sequenced, and expressed in E. coli. See Barbour, et al., Rev. Inf. Dis. 11(6): S1470-74 (1989); Bergstrom, et al., Mol. Microbiol. 3:479-86 (1989).
The pC (24 kd) protein of B. burgdorferi is similar to ospA and B in some respects. It, too is a lipoprotein and exhibits molecular weight and serological heterogeneity and is exposed on the cell-surface (it is available on the cell-surface to bind agglutinating antibody and cell-associated pC is susceptible to digestion by proteases). Strains expressing pC protein are common in Europe. Between 40% to 50 % of the 28 European isolates tested by Wilske, et al., N.Y. Acad. Sci. 539:126-43 (1988), were positive for the pC protein, although this may be an underestimate because pC expression is subject to fluctuations.
Other B. burgdorferi antigens include the outer-surface protein found in the 60 kd region, Barbour, et al., supra; the flagellar structural protein in the 41 kd region, Gassmann, et al., Nucleic Acids Res. 17:3590 (1989); the protein of the 39 kd region, Simpson, et al., J. Clin. Micro. 28: 1329-37 (1990); and an approximately 94 kd protein, Fuchs, et al., FOURTH INTERNATIONAL CONFERENCE ON LYME BORRELIOSIS (1990).
Various purification methods have been used in preparing antigens for further study and characterization in this context. For instance, Wilske, et al., Zbl. Bakt. Hyg. 263:92-102 (1986), subjected whole Borreliae to a SDS-PAGE regimen in which the proteins were denatured by heat and exposure to the detergent sodium dodecyl sulphate (SDS) and 2-mercaptoethanol. Hansen, et al., J. Clin. Microbiol. 26(2): 338-46 (1988), disclosed the purification of B. burgdorferi flagellum. World Patent Application No. 90/04411 by Bergstrom, et al. teaches a non-denaturing method for partially purifying fractions of Borrelia burgdorferi.
Studies have also focused on the preparation and characterization of various antigens for purposes of developing diagnostic tests. Thus, a diagnostic procedure for detecting B. burgdorferi indirectly, by assaying for specific antibody production in response to infection, is disclosed in the aforementioned application of Bergstrom, et al., and in U.S. patent application Ser. No. 07/487,716 (Simpson & Schwan) (published Jul. 18, 1990).
Coleman, et al., J. Infect. Dis. 155:756-65 (1987), also disclose the production of a B. burgdorferi fractions by treating whole spirochaetes with denaturing SDS detergent, thereby to obtain a protoplasmic cylinder (the bacterium stripped of protein coat) fraction which, upon further treatment, can be employed as an antigen.
Wilske, et al., FOURTH INTERNATIONAL CONFERENCE ON LYME BORRELIOSIS (1990), report identifying immunodominant Borreliae proteins which are said to be useful in diagnosing Lyme borreliosis. These investigators conclude that two proteins, pC and p100, may be particularly important to the extent that they provide an indication of early and late stages of the disease, respectively.
Although various antigens are known, protective efficacy cannot be predicted from an antigen's ability to elicit an immune response in the course of a natural or experimental infection. For instance, the 41kd flagellar induces an immune response but is not protective. See Simon, et al., Immunology Today 12:11-16 (1991). The 94 kd protein similarly fails to provide protection, as reported below in Example 3. In fact, applicants have observed that antigens which are protective are relatively rare. Consequently, a large part of the immune response will be to antigens which are not relevant for protection. Conversely, some potentially protective antigens may fail to elicit an adequate immune response. Thus, utility as a vaccine constituent cannot be inferred from the ability of an antigen to elicit an antibody response.
Therefore, a need exists for continued research in the development of a suitable vaccine against Lyme borreliosis. Of interest in this regard is U.S. Pat. No. 4,721,617 (Johnson), which discloses a vaccine against Lyme borreliosis comprised of whole B. burgdorferi cells which had been inactivated by lyophilization. Based on recovery of the pathogen from kidney or spleen, Johnson demonstrates a dose-dependent reduction in the susceptibility of immunized hamsters to infection by a virulent B. burgdorferi strain. The effect was short-lived, however, and animals challenged 90 days post-vaccination were incompletely protected.
European Patent Application No. 418827 (Simon, et al.) describes a vaccine against B. burgdorferi, especially strains B31 or ZS7, comprised of monoclonal antibodies which recognize the 31 kD ospA protein. According to the aforementioned European application of Simon, et al., passive immunization of SCID-mice with these antibodies inhibits the development of Borrelia-induced symptoms. (Protection is defined in terms of resistance to infection and to the development of arthritis.) The European application also discloses expression in E. coli of a recombinant .beta.-galactosidase/ospA fusion protein. The disclosed monoclonal antibodies are raised by immunization with whole bacterial cell or with the recombinant antigenic proteins.
Fikrig, et al., Science 250:553-56 (1990), document the passive protection of mice (C3H/HeJ) with polyclonal sera to killed B. burgdorferi or to E.coli expressing ospA, or with an ospA-specific monoclonal antibody. The investigators also show that mice were actively protected upon immunization with a purified, recombinant ospA/glutathione S-transferase fusion protein. Protection was measured in terms of the immunogen's ability to prevent infection or to abrogate the histopathological manifestations of the disease.
Bergstrom, et al. (WO 90/04411), also suggest the possibility that immunogenically active B. burgdorferi fractions could be used in vaccines. No data are provided, however, to demonstrate either the immunogenicity or the protective efficacy of the disclosed fractions.